The Medicinal Chemistry Core will support Projects 2 and 3 with synthetic and medicinal chemistry. Projects 2 and 3 will collaborate with the HTS Core to find small molecules that disrupt or stabilize complexes involving low-complexity sequences (Project 2) and promote cell cycle arrest through suppression of EWSR1-FLI1 (Project 3). Aim 1 of the Med Chem Core will be to validate these initial hits. In particular, the Chemistry Core will evaluate initial hits to select the most promising compounds from both screens with regard to chemical reactivity, physicochemical characteristics and synthetic accessibility. We will then initiate medicinal chemistry campaigns to prepare 20-30 analogs of ~5 of the most attractive scaffolds. Analogs of each scaffold will be synthesized and then evaluated by the HTS core, Projects 2 and 3 and the PK/PD Core to select hit series that display a) synthetic accessibility to allow a robust medicinal chemistry program (see Aim 2), b) an initial structure-activity relationship consistent with engagement of a discrete protein target, and c) desirable pharmacokinetic and physicochemical properties. Aim 2 is to optimize initial hits from Projects 2 and 3 into validated lead compounds. Aim 1 will provide validated hits from two HTS experiments. This aim optimizes these initial hits into advanced lead compounds. Specifically, in collaboration with the HTS core, Projects 2 and 3 and the Pharmacology core, we will initiate a robust medicinal chemistry effort to optimize 2-4 of the most promising chemical scaffolds from each screen. With Project 2 (modulators of TAF15-RNA Pol II interaction), we will optimize compounds to provide chemical tools to interrogate the molecular basis of protein-protein interactions associated with low-complexity sequences. With Project 3 (EWSR1-FLI1 inhibition), we will aim to identify one or more compounds with 100 nM activity in cell culture, ADME characteristics suitable for in vivo evaluation, and efficacy using in vivo rodent models of human cancer. Moreover, optimized compounds will show no toxicity in vivo at >10X the therapeutic dose to allow for in vivo proof-of-concept studies. Aim 3 will entail target-oriented synthesis in support of Projects 2, 3, and the PK/PD Core. Medicinal chemistry efforts in support of Project 3 (Aim 2) will result in the discovery of optimized small molecules for suppression of EWSR1-FLI1-driven cell proliferation. The Chemistry Core will develop scalable syntheses of these substances to provide multi-gram quantities sufficient for animal testing of their metabolic profile, toxicity and efficacy. Furthermore, Projects 2 and 3 will require probe molecules for target identification and localization studies including radio-labeled compounds, dye- or biotin-conjugated analogs, and/or photo-crosslinking reagents. Building on extensive experience preparing these types of reagents in collaborations with two of the PI's (McKnight and Nijhawan), the Chemistry Core will design and synthesize suitable probes.